Thioredoxins are small (about 12 kDa) thermostable proteins with catalytically active disulfide groups. This class of proteins has been found in virtually all organisms, and has been implicated in myriad biochemical pathways (Buchanan et al., 1994). The active site of thioredoxin has two redox-active cysteine residues in a highly conserved amino acid sequence; when oxidized, these cysteines form a disulfide bridge (xe2x80x94Sxe2x80x94Sxe2x80x94) that can be reduced to the sulfhydryl (xe2x80x94SH) level through a variety of specific reactions. In physiological systems, this reduction may be accomplished by reduced ferredoxin, NADPH, or other associated thioredoxin-reducing agents. The reduced form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.
Generally only one kind of thioredoxin is found in bacterial or animal cells. In contrast, photosynthetic organisms have three distinct types of thioredoxin. Chloroplasts contain a ferredoxin/thioredoxin system comprised of ferredoxin, ferredoxin-thioredoxin reductase and thioredoxins f and m, which function in the light regulation of photosynthetic enzymes (Buchanan, 1991; Scheibe, 1991; Vogt et al, 1986). The other thioredoxin enzyme system is analogous to that established for animals and most microorganisms, in which thioredoxin (h-type in plants) is reduced by NADPH and NADPH-thioredoxin reductase (NTR) (Johnson et al., 1987a; Florencio et al., 1988; Suske et al., 1979). The reduction of thioredoxin h by this system can be illustrated by the following equation: 
Some plant species contain a family of closely related thioredoxin h proteins, which probably perform different physiological functions. Specific plants in which multiple thioredoxin h proteins have been found include spinach (Florencio et al., 1988), wheat (Johnson et a., 1987), rice (Ishiwatari et al., 1995), and Arabidopsis (Rivera-Madrid et al., 1995). The type-h thioredoxin was discovered considerably after the discovery of the m and f types, and because of this much less is known about this cytosolic thioredoxin and its physiological functions. Considerable work is currently directed toward studying thioredoxin h proteins (Besse and Buchanan, 1997).
Thioredoxin h is widely distributed in plant tissues and exists in mitochondria, endoplasmic reticulum (ER) and the cytosol (Bodenstein-Lang et al., 1989; Marcus et al., 1991; Vogt et al. 1986). Plant thioredoxin h is involved in a wide variety of biological functions. Thioredoxin h functions in the reduction of intramolecular disulfide bridges of a variety of low molecular-weight, cystine-rich proteins, including thionins (Johnson et al., 1987b), protease inhibitors and chloroform/methanol-soluble proteins (CM proteins) (Kobrehel et al, 1991). It is likely that cytoplasmic thioredoxins participate in developmental processes: for example thioredoxin h has been shown to function as a signal to enhance metabolic processes during germination and seedling development (Kobrehel et al., 1992; Lozano et al., 1996; Besse et al., 1996). Thioredoxin h has also been demonstrated to be involved in self-incompatibility in Phalaris coerulescens (Li et al., 1995) and Brassica napus (Bower et al., 1996). Several functions have been hypothesized for rice thioredoxin h, which is believed to be involved in translocation in sieve tubes (Ishiwatari et al., 1995).
Uses of thioredoxin include incorporation into hair care products (U.S. Pat. No. 4,935,231) and neutralization of certain venoms and toxins (see U.S. Pat. No. 5,792,506). Recent research into thioredoxin activity has also focused on harnessing the reducing power of this protein for food technology. For example, U.S. Pat. No. 5,792,506 to Buchanan (Neutralization of Food Allergens by Thioredoxin), and Buchanan et al. (1998) describe the use of thioredoxin to reduce the allergenicity of foods through thioredoxin-mediated reduction of intramolecular disulfide bonds found in various allergenic food proteins (e.g., in milk, soya and wheat proteins) (Buchanan et al, 1997; del Val et al., 1999). In addition, it has been shown that reduction of disulfide protein allergens in wheat and milk by thioredoxin decreases their allergenicity (Buchanan et al., 1997; del Val et al., 1999). Thioredoxin treatment also increases the digestibility of the major allergen of milk (xcex2-lactoglobulin) (del Val et al., 1999), as well as other disulfide proteins (Lozano et al., 1994; Jiao et al., 1992).
Thioredoxin h has been shown to be useful as a food additive to enhance the baking qualities of cereal flour (Bright et al., 1983). For example, improvement in dough strength and bread quality properties of poor-quality wheat flour results from the addition of thioredoxin (Wong et al., 1993; Kobrehel et al., 1994). This has been attributable to the thioredoxin-catalyzed reduction of intramolecular disulfide bonds in the flour proteins, specifically the glutenins, resulting in the formation of new intermolecular disulfide bonds (Besse and Buchanan, 1997). Thus, the addition of exogenous thioredoxin promotes the formation of a protein network that produces flour with enhanced baking quality. Kobrehel et at, (1994) have observed that the addition of thioredoxin h to flour of non-glutenous cereals such as rice, maize and sorghum promotes the formation of a dough-like product. Hence, the addition of exogenous thioredoxin may be used to produce baking dough from non-glutenous cereals.
cDNA clones encoding thioredoxin h have been isolated from a number of plant species, including
Arabidopsis thaliana (Rivera-Madrid et al., 1993; Rivera-Madrid et al., 1995), Nicotiana tabacum (Marty and Meyer, 1991; Brugidou et al., 1993), Oryza sativa (Ishiwatari et al., 1995), Brassica napus (Bower et al, 1996), Glycine max (Shi and Bhattacharyya, 1996), and Triticum aestivum (Gautier et al., 1998).
Thioredoxin and NTR were first characterized in Escherichia coli as the hydrogen donor system for ribonucleotide reductase (Laurent et al., 1964 Moore et al., 1964) The E. coli NTR gene has been isolated (Russel and Model, 1988) and the three-dimensional structure of the protein has been analyzed (Kuriyan et al., 1991). Some other NTR genes have been isolated and sequenced from bacteria, fungi and mammals. Recently, Jacquot et al. (1994) have reported a successful isolation and sequencing of two cDNAs encoding the plant Arabidopsis thaliana NTRs. The subsequent expression of the recombinant A. thaliana NTR protein in E. coli cells (Jacquot et al., 1994) and its first eukaryotic structure (Dai et al., 1996) have also been reported.
Thioredoxin and NTR were first characterized in Escherichia coli as the hydrogen donor system for ribonucleotide reductase (Laurent et al, 1964; Moore et al., 1964) The E. coli NTR gene has been isolated (Russel and Model, 1988) and the three-dimensional structure of the protein has been analyzed (Kuniyan et al., 1991). Some other NTR genes have been isolated and sequenced from bacteria, fungi, and mammals. Recently, Jacquot et al. (1994) have reported a successful isolation and sequencing of two cDNAs encoding the plant Arabidopsis thaliana NTRs. The subsequent expression of the recombinant A. thaliana NTR protein in E. coli cells (Jacquot et al., 1994) and its first eukaryotic structure (Dai et al., 1996) have also been reported.
Here we report isolated nucleic acids encoding the barley genes for thioredoxin h and NADP-thioredoxin reductase; isolated barley thioredoxin h and NADP-thioredoxin reductase proteins, and methods of use.
The invention provides isolated nucleic acids encoding barley thioredoxin and NADP-thioredoxin reductase proteins and methods of use.
In other aspect the invention provides expression vectors comprising nucleic acids encoding barley thioredoxin and NADP-thioredoxin and transformed host cells. Accordingly, the invention provides methods of expressing an isolated barley thioredoxin and NADP-thioredoxin reductase polypeptides.
In a further aspect the invention provides transgenic plants comprising the expression vectors. In a preferred embodiment, the transgenic plants overexpress barley thioredoxin and NADP-thioredoxin reductase polypeptides. The polypeptides of the invention, expressed in a transgenic plant either alone or in combination alters the redox status of a plant in comparison to a nontransgenic plant of the same species. In a preferred embodiment, the expressed polypeptide transgene alters the redox status of a seed or grain, thereby altering the biochemical and biological properties of a seed or grain. The seed or grain provides advantages in increased germination efficiency, decreased allergenicity, increased protein solubility, increased digestibility.
In another aspect, the invention provides methods of expressing a barley thioredoxin or NADP-thioredoxin reductase polypeptides.
In yet another aspect, the invention provides of expressing a barley thioredoxin or NADP-thioredoxin reductase polypeptide. Accordingly, the invention provides isolated barley thioredoxin or NADP-thioredoxin reductase polypeptides.
In still yet another aspect the invention provides methods of identifying a bioactive agent that binds and preferably reduces a biological activity of a barley thioredoxin or NADP-thioredoxin reductase polypeptide.